RESUMO
The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.
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Actinas , Peixe-Zebra , Animais , Humanos , Actinas/metabolismo , Vimentina/genética , Vimentina/metabolismo , Peixe-Zebra/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Movimento Celular/fisiologia , Proteínas de Transporte/metabolismoRESUMO
Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape and cytoskeleton of these lateral protrusions and distinguish them from cytonemes, filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics, and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1 (also known as WASF1): misexpression of mutant WAVE1 attenuated protrusion and generated a round-ended apical endfoot morphology. However, this did not alter apico-basal cell polarity or tissue integrity. During normal neuronal delamination, lateral protrusions were withdrawn, but precocious protrusion loss induced by mutant WAVE1 was insufficient to trigger neurogenesis. This study uncovers a new form of cell-cell contact within the developing neuroepithelium, regulation of which prefigures neuronal delamination. This article has an associated First Person interview with the first author of the paper.
Assuntos
Actinas , Células Neuroepiteliais , Actinas/metabolismo , Citoesqueleto/metabolismo , Humanos , Células Neuroepiteliais/metabolismo , Neurogênese , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
OBJECTIVES: Sickle cell disease (SCD) is an inherited disorder that causes lifelong complications, substantially impacting the physical and emotional well-being of patients and their caregivers. Studies investigating the effects of SCD on quality of life (QOL) are often limited to individual countries, lack SCD-specific QOL questionnaires, and exclude the caregiver experience. The SHAPE survey aimed to broaden the understanding of the global burden of SCD on patients and their caregivers and to capture the viewpoint of healthcare providers (HCPs). METHODS: A total of 919 patients, 207 caregivers, and 219 HCPs from 10, 9, and 8 countries, respectively, answered a series of closed-ended questions about their experiences with SCD. RESULTS: The symptoms most frequently reported by patients were fatigue/tiredness (84%) and pain/vaso-occlusive crises (71%). Patients' fatigue/tiredness had one of the greatest impacts on both patients' and caregivers' QOL. On average, patients and caregivers reported missing 7.5 days and 5.0 days per month, respectively, of school or work. HCPs reported a need for effective tools to treat fatigue/tiredness and a desire for more support to educate patients on long-term SCD-related health risks. CONCLUSIONS: The multifaceted challenges identified using the SHAPE survey highlight the global need to improve both patient and caregiver QOL.
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Anemia Falciforme , Cuidadores , Pessoal de Saúde , Qualidade de Vida , Humanos , Anemia Falciforme/psicologia , Anemia Falciforme/epidemiologia , Anemia Falciforme/terapia , Cuidadores/psicologia , Adulto , Pessoal de Saúde/psicologia , Adolescente , Masculino , Feminino , Inquéritos e Questionários , Adulto Jovem , Efeitos Psicossociais da Doença , Conhecimentos, Atitudes e Prática em Saúde , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Teenage pregnancies are a global concern. Malawi is one of the countries with the highest teenage pregnancy rates despite government efforts to reverse the situation and yet studies on determinants of teenage pregnancy are rare with some factors remaining unexplored. Therefore, this study aimed to identify factors associated with teenage pregnancies in Malawi. METHODS: This was a community-based case-control study that used secondary data from the 2015-16 Malawi Demographic and Health Survey from all 28 districts of Malawi. The study population comprised women aged 20-24 who participated in the survey. The study ran from September 2021 to October 2022 and used a sample size of 3,435 participants who were all women aged 20-24 in the dataset who met the inclusion criteria. Data were analysed using Stata 16 software. Logistic regression analyses were used to determine factors. Variables with a P value of < 0.1 in the univariable analysis were included in the multivariable analyses, where statistical significance was obtained at a P value < 0. 05. RESULTS: Data on 3435 participants were analysed. In multivariable analyses: no teenage marriage (AOR 0.13); secondary education (AOR 0.26); higher education (AOR 0.39); richest category of wealth index (AOR 0.51), use of contraception (AOR 3.08), domestic violence by father or mother (AOR 0.37) were found to be significant factors. CONCLUSION: This study identified determinants of teenage pregnancy. The government has to sustain and expand initiatives that increase protection from teenage pregnancy, reinforce the implementation of amended marriage legislation, introduce policies to improve the socioeconomic status of vulnerable girls and increase contraceptive use among adolescent girls before their first pregnancy. Further research is also recommended to resolve inconclusive results.
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Gravidez na Adolescência , Humanos , Feminino , Gravidez na Adolescência/estatística & dados numéricos , Malaui , Gravidez , Estudos de Casos e Controles , Adolescente , Adulto Jovem , Comportamento Contraceptivo/estatística & dados numéricos , Casamento/estatística & dados numéricos , Fatores Socioeconômicos , Fatores de Risco , Escolaridade , Anticoncepção/estatística & dados numéricos , Modelos LogísticosRESUMO
Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.
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Anticorpos Biespecíficos , Leucemia Mieloide Aguda , Animais , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Proteínas de Checkpoint Imunológico/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos TRESUMO
PURPOSE: The Curaçao criteria are well-established diagnostic criteria for hereditary hemorrhagic telangiectasia (HHT), but they lack details regarding a predictive presentation of epistaxis and telangiectasias. This study collects and compares data in HHT and population cohorts to inform the application of these criteria. METHODS: In-person interviews regarding epistaxis and targeted examination for telangiectases in a general population cohort (n = 204) and an HHT cohort (n = 432) were conducted. RESULTS: Frequency of epistaxis, rather than intensity or duration, was the best discriminator of HHT. A cutoff of ≥4 nosebleeds per year alone yielded a diagnostic sensitivity of 97%, and specificity of 84%. The mean number of telangiectases at the sites investigated was 0.4 in the general population cohort and 26.5 in the HHT cohort. The most distinctive sites for telangiectases in HHT were lips and palmar fingers, whereas telangiectases of the face and dorsum of the hand were comparable in both cohorts. CONCLUSION: We propose that the Curaçao criteria be modified to include the following cutoffs: (1) epistaxis frequency of ≥4 nosebleeds per year and (2) telangiectasia count of at least 2 in characteristic locations (palmar aspect of fingers, lips, and oral cavity), and that cutaneous telangiectases at other sites not be considered relevant for diagnostic purposes.
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Telangiectasia Hemorrágica Hereditária , Telangiectasia , Humanos , Telangiectasia Hemorrágica Hereditária/complicações , Telangiectasia Hemorrágica Hereditária/diagnóstico , Telangiectasia Hemorrágica Hereditária/epidemiologia , Epistaxe/epidemiologia , Epistaxe/etiologia , Epistaxe/diagnóstico , Curaçao , Telangiectasia/diagnóstico , Telangiectasia/epidemiologia , PacientesRESUMO
The mechanisms of particle-induced pathogenesis in the lung remain poorly understood. Neutrophilic inflammation and oxidative stress in the lung are hallmarks of toxicity. Some investigators have postulated that oxidative stress from particle surface reactive oxygen species (psROS) on the dust produces the toxicopathology in the lungs of dust-exposed animals. This postulate was tested concurrently with the studies to elucidate the toxicity of lunar dust (LD), which is believed to contain psROS due to high-speed micrometeoroid bombardment that fractured and pulverized lunar surface regolith. Results from studies of rats intratracheally instilled (ITI) with three LDs (prepared from an Apollo-14 lunar regolith), which differed 14-fold in levels of psROS, and two toxicity reference dusts (TiO2 and quartz) indicated that psROS had no significant contribution to the dusts' toxicity in the lung. Reported here are results of further investigations by the LD toxicity study team on the toxicological role of oxidants in alveolar neutrophils that were harvested from rats in the 5-dust ITI study and from rats that were exposed to airborne LD for 4 weeks. The oxidants per neutrophils and all neutrophils increased with dose, exposure time and dust's cytotoxicity. The results suggest that alveolar neutrophils play a critical role in particle-induced injury and toxicity in the lung of dust-exposed animals. Based on these results, we propose an adverse outcome pathway (AOP) for particle-associated lung disease that centers on the crucial role of alveolar neutrophil-derived oxidant species. A critical review of the toxicology literature on particle exposure and lung disease further supports a neutrophil-centric mechanism in the pathogenesis of lung disease and may explain previously reported animal species differences in responses to poorly soluble particles. Key findings from the toxicology literature indicate that (1) after exposures to the same dust at the same amount, rats have more alveolar neutrophils than hamsters; hamsters clear more particles from their lungs, consequently contributing to fewer neutrophils and less severe lung lesions; (2) rats exposed to nano-sized TiO2 have more neutrophils and more severe lesions in their lungs than rats exposed to the same mass-concentration of micron-sized TiO2; nano-sized dust has a greater number of particles and a larger total particle-cell contact surface area than the same mass of micron-sized dust, which triggers more alveolar epithelial cells (AECs) to synthesize and release more cytokines that recruit a greater number of neutrophils leading to more severe lesions. Thus, we postulate that, during chronic dust exposure, particle-inflicted AECs persistently release cytokines, which recruit neutrophils and activate them to produce oxidants resulting in a prolonged continuous source of endogenous oxidative stress that leads to lung toxicity. This neutrophil-driven lung pathogenesis explains why dust exposure induces more severe lesions in rats than hamsters; why, on a mass-dose basis, nano-sized dusts are more toxic than the micron-sized dusts; why lung lesions progress with time; and why dose-response curves of particle toxicity exhibit a hockey stick like shape with a threshold. The neutrophil centric AOP for particle-induced lung disease has implications for risk assessment of human exposures to dust particles and environmental particulate matter.
Assuntos
Poeira , Pneumopatias , Cricetinae , Ratos , Humanos , Animais , Neutrófilos/patologia , Pulmão , Citocinas/toxicidade , Oxidantes/toxicidade , Tamanho da PartículaRESUMO
INTRODUCTION: In carefully selected patients with medically refractory epilepsy, disconnective hemispherotomy can result in significant seizure freedom; however, incomplete disconnection can result in ongoing seizures and poses a significant challenge. Completion hemispherotomy provides an opportunity to finish the disconnection. We describe the use of magnetic resonance-guided laser interstitial thermal ablation (MRgLITT) for completion hemispherotomy. METHODS: Patients treated with completion hemispherotomy using MRgLITT at our institution were identified. Procedural and seizure outcomes were evaluated retrospectively. RESULTS: Five patients (3 males) underwent six MRgLITT procedures (one child treated twice) for completion hemispherotomy at a median age of 6 years (range 1.8-12.9). Two children had hemimegalencephaly, two had Rasmussen encephalitis, and one had polymicrogyria. All five children had persistent seizures likely secondary to incomplete disconnection after their functional hemispherotomy. The mean time from open hemispherotomy to MRgLITT was 569.5 ± 272.4 days (median 424, range 342-1,095). One patient underwent stereoelectroencephalography before MRgLITT. The mean number of ablation targets was 2.3 ± 0.47 (median 2, range 2-3). The mean length of the procedure was 373 min ± 68.9 (median 374, range 246-475). Four of the five patients were afforded improvement in their neurocognitive functioning and speech performance after ablation, with mean daily seizure frequency at 1 year of 1.03 ± 1.98 (median 0, range 0-5). Two patients achieved Engel Class I outcomes at 1 year after ablation, one was Engel Class III, and two were Engel Class IV. The mean follow-up time was 646.8 ± 179.5 days (median 634, range 384-918). No MRgLITT-related complications occurred. Delayed retreatment (>1 year) occurred in three patients: one child underwent redo ablation and two underwent anatomic hemispherectomy. CONCLUSION: We have demonstrated the feasibility of a minimally invasive approach for completion hemispherotomy using MRgLITT. Delayed retreatment was needed in three patients; thus, further study of this technique with comparison to other surgical techniques is warranted.
Assuntos
Epilepsia Resistente a Medicamentos , Hemisferectomia , Terapia a Laser , Criança , Masculino , Humanos , Lactente , Pré-Escolar , Estudos Retrospectivos , Resultado do Tratamento , Imageamento por Ressonância Magnética/métodos , Epilepsia Resistente a Medicamentos/diagnóstico por imagem , Epilepsia Resistente a Medicamentos/cirurgia , Convulsões/cirurgia , Terapia a Laser/efeitos adversos , Hemisferectomia/efeitos adversos , Hemisferectomia/métodos , Espectroscopia de Ressonância Magnética/efeitos adversosRESUMO
Asthma is a heterogeneous disease, with multiple underlying inflammatory pathways and structural airway abnormalities that impact disease persistence and severity. Recent progress has been made in developing targeted asthma therapeutics, especially for subjects with eosinophilic asthma. However, there is an unmet need for new approaches to treat patients with severe and exacerbation-prone asthma, who contribute disproportionately to disease burden. Extensive deep phenotyping has revealed the heterogeneous nature of severe asthma and identified distinct disease subtypes. A current challenge in the field is to translate new and emerging knowledge about different pathobiologic mechanisms in asthma into patient-specific therapies, with the ultimate goal of modifying the natural history of disease. Here, we describe the Precision Interventions for Severe and/or Exacerbation-Prone Asthma (PrecISE) Network, a groundbreaking collaborative effort of asthma researchers and biostatisticians from around the United States. The PrecISE Network was designed to conduct phase II/proof-of-concept clinical trials of precision interventions in the population with severe asthma, and is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health. Using an innovative adaptive platform trial design, the PrecISE Network will evaluate up to 6 interventions simultaneously in biomarker-defined subgroups of subjects. We review the development and organizational structure of the PrecISE Network, and choice of interventions being studied. We hope that the PrecISE Network will enhance our understanding of asthma subtypes and accelerate the development of therapeutics for severe asthma.
Assuntos
Asma/tratamento farmacológico , Medicina de Precisão , Comitês Consultivos , Asma/diagnóstico , Biomarcadores , Protocolos Clínicos , Ensaios Clínicos Fase II como Assunto , Humanos , Projetos de Pesquisa , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios XRESUMO
T cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response, which is hypothesized to depend on T cells combining stimuli from multiple antigen-presenting cell interactions into a more potent response. To quantify the capacity for T cells to accomplish this, we have developed an antigen receptor that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling. We observe limited persistence within the T-cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 min, and directly show sustained proximal receptor signaling is required to maintain gene transcription. T cells thus primarily accumulate the outputs of gene expression rather than integrate discrete intracellular signals. Engineering optical control in a clinically relevant chimeric antigen receptor (CAR), we show that this limited signal persistence can be exploited to increase CAR-T cell activation threefold using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks.
Assuntos
Optogenética/métodos , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Cálcio/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Células Jurkat , Ativação Linfocitária , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Transdução de SinaisRESUMO
The international Sickle Cell World Assessment Survey (SWAY) reported a high impact of sickle cell disease (SCD) on patients' daily lives globally. In this study, we analyzed whether the reported burden differed between patients from the USA (n = 384) and other high-income (HI; n = 820) or low- to middle-income (LMI; n = 941) countries. We assessed symptoms and complications, incidence/management of vaso-occlusive crises (VOCs), treatment utilization/satisfaction, and the impact of SCD on education/employment. Certain symptoms (bone aches, insomnia, and joint stiffness) and complications (swollen/painful fingers/toes, gallstones, vision problems, blood clots, and asthma) were reported proportionally more by patients in the USA than in the HI/LMI countries. Self-reported VOCs were more common (mean [SD]: 7.1 [5.7] vs. 5.5 [8.9] and 4.4 [4.6] in the previous 12 months) and were managed more often by hospitalization (52% vs. 24% and 32%) in the USA than the HI and LMI countries. A higher proportion of patients from the USA than the HI/LMI countries reported a negative impact of SCD on their employment/schooling. Although high overall satisfaction with current treatments was reported globally, most patients indicated a strong desire for alternative pain medications. There are likely several reasons for the relatively high patient-reported burden in the USA group compared with the HI/LMI countries, including an older population and differences in newborn screening programs and pediatric/adult transition of care. It is clear that there is an urgent need for improved understanding and management of SCD globally, not just in the USA.
Assuntos
Anemia Falciforme , Países em Desenvolvimento , Adulto , Anemia Falciforme/complicações , Anemia Falciforme/epidemiologia , Anemia Falciforme/terapia , Criança , Efeitos Psicossociais da Doença , Emprego , Humanos , Recém-Nascido , Dor/tratamento farmacológicoRESUMO
Humans will set foot on the Moon again soon. The lunar dust (LD) is potentially reactive and could pose an inhalation hazard to lunar explorers. We elucidated LD toxicity and investigated the toxicological impact of particle surface reactivity (SR) using three LDs, quartz, and TiO2. We first isolated the respirable-size-fraction of an Apollo-14 regolith and ground two coarser samples to produce fine LDs with increased SR. SR measurements of these five respirable-sized dusts, determined by their in-vitro ability to generate hydroxyl radicals (â¢OH), showed that ground LDs > unground LD ≥ TiO2 ≥ quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5 mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13 weeks after the bolus instillation. All dusts caused dose-dependent-increases in pulmonary lesions and toxicity biomarkers. The three LDs, which possessed mineral compositions/properties similar to Arizona volcanic ash, were moderately toxic. Despite a 14-fold â¢OH difference among these three LDs, their toxicities were indistinguishable. Quartz produced the lowest â¢OH amount but showed the greatest toxicity. Our results showed no correlation between the toxicity of mineral dusts and their ability to generate free radicals. We also showed that the amounts of oxidants per neutrophil increased with doses, time and the cytotoxicity of the dusts in the lung, which supports our postulation that dust-elicited neutrophilia is the major persistent source of oxidative stress. These results and the discussion of the crucial roles of the short-lived, continuously replenished neutrophils in dust-induced pathogenesis are presented.
Assuntos
Poeira , Pneumopatias , Animais , Biomarcadores , Poeira/análise , Pneumopatias/induzido quimicamente , Lua , Oxidantes/toxicidade , Quartzo/toxicidade , Ratos , Dióxido de Silício/toxicidade , TitânioRESUMO
INTRODUCTION AND AIM OF STUDY: Tranexamic acid has been found to be effective in reducing peri-operative blood loss and is widely used across surgical specialties including orthopaedic surgery. However, there is still no consensus on the most appropriate and effective dose regimen. This study therefore compared the efficacy of single versus double dose regimens in patients that had interlocking intramedullary nailing by assessing the volume of drain output with the objective of determining the more effective regimen. METHODS: The study was a multicenter prospective study amongst adult patients who had interlocking intramedullary nailing for femoral nonunions. Following ethical approval, consent was obtained from eligible patients who were randomly assigned into two study groups. Group A patients had single pre-incision tranexamic acid bolus of one gram while group B patients had a second (repeat) one gram bolus (at the completion of wound closure). The volume of drain output at 48 h postop was the primary outcome measure and data collection was via an online data collection form linked to the google drive of the principal investigator. The mean of the drain output of the two groups was compared for statistical significance. RESULTS: A total of 61 patients participated in the study with 30 patients in group A and 31 patients in group B. The demographic data and duration of fracture were comparable in the two groups. Group A had a mean drain volume of 274.80 ml (± 103.93 ml) while group B had a mean of 187.94 ml (± 41.95 ml) and the difference was found to be statistically significant. (P, 0.000). CONCLUSION: The findings suggest that double dose perioperative tranexamic acid regimen is superior to single-dose peri-operative tranexamic acid regimen in reducing post-operative blood loss in patients undergoing interlocking intramedullary nailing for femoral nonunions.
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Perda Sanguínea Cirúrgica , Fraturas do Fêmur , Fixação Intramedular de Fraturas , Ácido Tranexâmico , Adulto , Perda Sanguínea Cirúrgica/prevenção & controle , Fraturas do Fêmur/cirurgia , Humanos , Estudos Prospectivos , Ácido Tranexâmico/administração & dosagem , Ácido Tranexâmico/uso terapêutico , Resultado do TratamentoRESUMO
Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.
Assuntos
Mama , Células Epiteliais , Humanos , Vimentina/metabolismo , Mama/metabolismo , Células Epiteliais/metabolismo , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Linhagem Celular Tumoral , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas de Homeodomínio/genéticaRESUMO
Sickle cell disease (SCD) is a genetic disorder, characterized by hemolytic anemia and vaso-occlusive crises (VOCs). Data on the global SCD impact on quality of life (QoL) from the patient viewpoint are limited. The international Sickle Cell World Assessment Survey (SWAY) aimed to provide insights into patient-reported impact of SCD on QoL. This cross-sectional survey of SCD patients enrolled by healthcare professionals and advocacy groups assessed disease impact on daily life, education and work, symptoms, treatment goals, and disease management. Opinions were captured using a Likert scale of 1-7 for some questions; 5-7 indicated "high severity/impact." Two thousand one hundred and forty five patients (mean age 24.7 years [standard deviation (SD) = 13.1], 39% ≤18 years, 52% female) were surveyed from 16 countries (six geographical regions). A substantial proportion of patients reported that SCD caused a high negative impact on emotions (60%) and school achievement (51%) and a reduction in work hours (53%). A mean of 5.3 VOCs (SD = 6.8) was reported over the 12 months prior to survey (median 3.0 [interquartile range 2.0-6.0]); 24% were managed at home and 76% required healthcare services. Other than VOCs, fatigue was the most commonly reported symptom in the month before survey (65%), graded "high severity" by 67% of patients. Depression and anxiety were reported by 39% and 38% of patients, respectively. The most common patient treatment goal was improving QoL (55%). Findings from SWAY reaffirm that SCD confers a significant burden on patients, epitomized by the high impact on patients' QoL and emotional wellbeing, and the high prevalence of self-reported VOCs and other symptoms.
Assuntos
Anemia Falciforme/psicologia , Atitude Frente a Saúde , Efeitos Psicossociais da Doença , Inquéritos Epidemiológicos , Qualidade de Vida , Atividades Cotidianas , Dor Aguda/epidemiologia , Dor Aguda/etiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Falciforme/complicações , Anemia Falciforme/epidemiologia , Ansiedade/etiologia , Criança , Estudos Transversais , Depressão/etiologia , Gerenciamento Clínico , Escolaridade , Emoções , Emprego/estatística & dados numéricos , Fadiga/epidemiologia , Fadiga/etiologia , Feminino , Cefaleia/epidemiologia , Cefaleia/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. METHODS: Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. RESULTS: Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45-/-/Csk-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. CONCLUSIONS: Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. MAJOR FINDINGS: Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. Video Abstract.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/imunologia , Tirosina/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Cinética , Camundongos Endogâmicos C57BL , Fosforilação , Conformação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/imunologia , Relação Estrutura-Atividade , Especificidade por Substrato , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.